Yiyi Fuzi Baijiang Decoction Alleviates Ulcerative Colitis Partly by Regulating TLR4-Mediated PI3K/Akt and NF-κB Pathways

Yiyi Fuzi Baijiang Decoction (YFBD), an ancient prescription developed by the ancient Chinese physician, Zhang Zhongjing, has shown remarkable effects in treating ulcerative colitis (UC). However, there are few studies on its mechanism. This study was designed to explore the potential mechanism of YFBD in treating UC. The principal ingredients of YFBD were analyzed using high-performance liquid chromatography (HPLC). Dextran sulfate sodium- (DSS-) induced mice and lipopolysaccharide- (LPS-) stimulated RAW264.7 cells were used in the study. The body weight and disease activity index (DAI) of mice were recorded and analyzed for 10 days. After sacrifice, the colonic tissues were harvested. The colon length was measured, and the histopathological changes were observed by hematoxylin and eosin staining. The levels of inflammatory cytokines in mice colons and RAW246.7 cells were determined by real-time quantitative PCR and immunofluorescence. The effects of YFBD on the TLR4-mediated PI3K/Akt and NF-κB pathways were determined by western blot analysis. HPLC identified five compounds in YFBD: chlorogenic acid, caffeic acid, benzoylmesaconine, benzoyl aconitine, and quercetin. YFBD alleviated weight loss, colon shortening, and colonic histopathological lesion in mice. Meanwhile, it decreased the DAI and histological score of mice with UC. In addition, YFBD remarkably decreased the levels of interleukin- (IL-) 6, IL-1β, and tumor necrosis factor (TNF)-α in the colons of DSS-induced mice and LPS-stimulated RAW246.7 cells. Furthermore, the expression of key proteins in TLR4-mediated PI3K/Akt and NF-κB pathways significantly decreased with YFBD treatment. In conclusion, YFBD had protective effects on mice with UC, which was in part related to its anti-inflammatory effects and downregulation of TLR4-mediated PI3K/Akt and NF-κB pathways.


Introduction
Ulcerative colitis (UC) is characterized by chronic nonspecific inflammation with recurring cycles of tissue destruction and repair. e highest incidence of UC was reported in Europe and North America (24.3 and 19.2 per 100,000, resp.) [1]. e morbidity follows an ascending trend, especially in newly industrialized countries [2][3][4]. Currently, drugs for UC, including aminosalicylates, corticosteroids, immunomodulators, and tumor necrosis factor (TNF)-α inhibitor, have achieved favorable efficacy in clinical practice. Sulfasalazine is one kind of traditional aminosalicylate products, which serves as first-line therapy on UC and is beneficial to most patients with mild-to-moderate, left-sided colitis [5]. However, these agents have limitations, including severe side effects, even tumorigenesis, and high costs [6]. erefore, more effective, less toxic, and cheaper drugs need to be developed to benefit patients with UC.
Traditional Chinese medicine (TCM) was demonstrated to have the therapeutic potential to treat UC [7,8]. Yiyi Fuzi Baijiang Decoction (YFBD), a formula originally presented in the Chinese medical classic Shang Han Lun, can be used to treat UC based on TCM theory. YFBD showed positive effects among patients [9,10]. It has three components: C. lacryma-jobi L. (Yi Ren), Radix Aconiti Lateralis Preparata (Fuzi), and Patrinia scabiosaefolia Fisch. (Bai Jiang Cao). C. lacryma-jobi L. could relieve the symptoms of UC by regulating the proinflammatory mediators and improving the microbial community structure in the gut [11,12]. Radix Aconiti Lateralis Preparata had far-ranging pharmacological activities; it displayed anti-inflammatory, antitumor, and immunoregulatory effects and influenced energy metabolism [13]. It also reduced inflammation in mice with UC partly by inhibiting the NF-kB pathway [14]. e extracts in P. scabiosaefolia Fisch. could reduce inflammatory responses in extensive models, such as UC, acute pancreatitis, and focal cerebral ischemia-reperfusion models [15]. Previous studies provided clues and evidence on the anti-inflammatory effects of YFBD. However, the underlying mechanism remains to be explored.
TLR4-mediated PI3K/Akt and NF-κB pathways closely relate to inflammatory responses of UC. TLR4, as a member of the Toll-like receptor family, can mediate inflammatory responses [16]. Previous studies confirmed that TLR4 expression was upregulated in patients with UC [17,18]. TLR4 recognized lipopolysaccharide (LPS) and initiated intracellular signaling, such as PI3K/Akt and NF-κB pathways [19][20][21][22]. e PI3K/Akt pathway was generally considered to be linked with the cellular processes of metabolism, proliferation, growth, and survival. However, current evidence suggests that the pathway participates in the pathogenesis of UC based on its extensive regulation of inflammation, apoptosis, and immune responses [23][24][25]. NF-κB is involved in gene expression of inflammatory cytokines [26]. Studies showed that persistent activation of NF-κB signaling could be detected in patients with UC and murine UC models [7,18]. erefore, it was inferred that TLR4-mediated PI3K/Akt and NF-κB pathways were critical to the development of UC and were probably promising therapeutic targets.
Consequently, considering the pharmacological study on the ingredients of YFBD and the mechanism of UC, it was hypothesized that YFBD could alleviate UC by controlling inflammation, which was partly realized by regulating TLR4mediated PI3K/Akt and NF-κB pathways. In the research, the levels of inflammatory cytokines and key proteins in these pathways were measured, and the possible mechanism behind YFBD treating UC was clarified for the first time.

Animals.
Fifty male C57BL/6 mice (age: 6 weeks; weight: 18-20 g) were purchased from Suzhou JOINN Clinical Co., Ltd. with license No. SYXK (Su) 2017-0043. ey were housed in a controlled environment at a temperature (24 ± 1°C) under a standard light-dark cycle with free access to food and drink. e animal research was conducted conforming to the protocol approved by the Institute of Animal Care Committee of Zhangjiagang TCM Hospital.

Grouping and Induction of UC.
e mice were randomly divided into five groups (n � 10): control group (sterile water), colitis group (2.5% DSS), SASP group (2.5% DSS + SASP 0.1 g/kg), YFBD low-dose group (2.5% DSS + YFBD 10 g/kg), and YFBD high-dose group (2.5% DSS + YFBD 20 g/kg). e experiment was started after seven days of adaptive feeding. e mice in the control group received sterile water for 10 days. e mice in the DSS, SASP, and YFBD groups were given 2.5% DSS for seven days to introduce colitis models, following which they were fed sterile water for three days. Meanwhile, the mice in treatment groups were orally administered with SASP (0.1 g/kg), YFBD (10 g/kg), and YFBD (20 g/kg) for 10 days. e mice in control and DSS groups were given the equal volume of distilled water by gavage. Animal equivalent doses were converted from human doses based on the body surface area [29]. After sacrifice, mice colons were cut into three sections for further research. e animal experimental design is shown in Figure 1(a).

Disease Activity Index and Histological Evaluation.
During drug administration and modeling, the body weight, stool characteristics, and bleeding were observed and recorded daily according to scoring system referred earlier [30]. e distal colonic tissues were fixed with 10% formalin, embedded in paraffin, sliced into four-µm thick sections, and mounted on microscope slides. e slices were tinted with hematoxylin and eosin (H&E) and then photographed under a microscope. e colonic pathology was scored based on the modified histology scoring system as described previously [31].

Cell
Culture and Treatment. RAW264.7 macrophages were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). e cells were cultured in highglucose DMEM (Hyclone, UT, USA) supplemented with 10% FBS (Biological Industries, Israel), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (NCM Biotech, Suzhou, China) in the presence of humidified 5% CO 2 at 37°C. e cells were seeded at 10 5 /mL for 24 h, followed by incubation with different concentrations of YFBD for 2 h, and then stimulated with LPS (1 μg/mL) for another 24 h.

Small Interfering RNA (siRNA) Transfection.
e TLR4 siRNA target sequence and negative control siRNA sequence were purchased from Santa Cruz Biotechnology (CA, USA). Briefly, RAW 264.7 cells were cultured in 6-well plates (5 × 10 5 cells/well). When the cell density reached 40-50%, NC siRNA or TLR4 siRNA were transfected into cells with Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's protocol. After a 24-hour transfection period, the cells were treated for 2 h with or without YFBD and then exposed to LPS (1 μg/ml) for 24 hours, followed by other analyses.

RNA Isolation and RT-qPCR Assay.
Total RNA was isolated from colon tissues and RAW 264.7 cells with TRIzol regent (Invitrogen, CA, USA). e RNA concentration was examined using a NanoDrop spectrophotometer ( ermo, MA, USA), and then RNA was reverse-transcribed to cDNA using an Applied Biosystems thermal cycler ( ermo, MA, USA). Afterwards, qRT-PCR was performed on a Light-Cycler 96 real-time PCR detection system (Roche, BW, Germany) following the instructions of SYBR Green ( ermo). Relative mRNA expression was calculated using the comparative Ct method (2 −△△Ct ). e primers were purchased from Sangon Biotech (Shanghai, China), and the sequences are listed in Table 2.
2.11. Immunofluorescence Staining. Immunofluorescence staining was performed on colon paraffin sections with 4 μm thickness. Briefly, the sections were boiled in citric acid buffer (Beyotime, Shanghai, China) for 20 min. After washing with PBS (Beyotime) three times, the sections were immersed in Triton X-100 (Beyotime) and subsequently blocked in 5% serum. Afterwards, they were incubated with primary antibodies at 4°C overnight. e colon sections were washed and coincubated with corresponding fluorescenceconjugated secondary antibodies in the dark, followed by staining with DAPI (Beyotime). Finally, the images of colon sections were captured under an epifluorescence microscope (Olympus, U-RFL-T, Japan).

Western Blot Analysis. A Protein Quantification Kit
(BCA Assay; ermo) was used to quantify the protein concentrations of extracts from colon tissues and cultured cells.

Statistical Analysis.
Each experiment was performed at least three times, and all data were expressed as mean ± SEM. e significance of differences was determined using oneway analysis of variance with Tukey's multiple comparison test using SPSS software 21.0. P values less than 0.05 indicated statistically significant differences.     Evidence-Based Complementary and Alternative Medicine

HPLC Analysis of the Main Constitutes in YFBD Extracts.
e chromatographs of the standard mixtures and the YFBD are shown in Figure 2. e contents of these five chemicals in YFBD (1 g/mL) were 106.2, 25.3, 62.5, 31.7, and 18.4 μg/mL for chlorogenic acid, caffeic acid, benzoylmesaconine, benzoyl aconitine, and quercetin, respectively.

YFBD Alleviated DSS-Induced
Colitis. Distinct body weight loss was observed from day 6 (P < 0.01), and the DAI score significantly increased on day 4 in the DSS group compared with the control group (P < 0.01). e body weight increased, and the DAI score dropped significantly in YFBD (20 g/kg) and SASP groups compared with the DSS group (P < 0.05 or 0.01; Figures 1(b) and 1(c)). Besides, DSS shortened the colon length significantly (P < 0.01; Figures 1(d) and 1(e)). However, the colon lengthened in the YFBD (10, 20 g/kg) and SASP groups compared with the DSS group (P < 0.01; Figures 1(d) and 1(e)). Furthermore, H&E staining showed that DSS administration induced severe colonic damage, such as mucosal ulceration and crypt damage along with leukocyte cell infiltration. However, treatment with YFBD (10, 20 g/kg) and SASP markedly attenuated colonic damage (Figure 1(f )). Moreover, the histological score increased appreciably in the DSS group (P < 0.01), while it declined in the YFBD (10, 20 g/kg) and SASP groups compared with the DSS group (P < 0.01; Figure 1(g)).

YFBD Decreased the Levels of Inflammatory Cytokines in
Mice with UC. A marked increase in mRNA levels of IL-6, IL-1β and TNF-α was observed after DSS induction in the control group (P < 0.01; Figures 3(a)-3(c)). However, the mRNA levels of the cytokines decreased in the YFBD (10 and 20 g/kg) and SASP groups compared with the DSS group (P < 0.05 or 0.01; Figures 3(a)-3(c)). Furthermore, immunofluorescence staining indicated that the fluorescence intensity of IL-6, IL-1β, and TNF-α was strong in the colonic tissues of DSS-induced mice, which were weakened in the YFBD (10 and 20 g/kg) and SASP groups (Figure 3(d)).

YFBD Decreased the Levels of Inflammatory Cytokines in
LPS-Stimulated RAW264.7 Cells. RAW264.7 cell models of experimental inflammation were used to further confirm the anti-inflammatory effects of YFBD [32,33]. Cytotoxicity analysis indicated that the concentrations of YFBD used in the experiment had no significant influence on cell viability (Figure 4(a)). e mRNA levels of IL-6, IL-1β, and TNF-α in RAW264.7 cells increased strikingly after induction with LPS (P < 0.01; Figures 4(b)-4(d)), while the cytokines declined in the YFBD groups (10, 50 μg/mL) compared with the DSS group (P < 0.05 or 0.01; Figures 4(b)-4(d)). Moreover, the study indicated that YFBD reduced the mRNA levels of IL-6, IL-1β, and TNF-α in a concentrationdependent manner.

YFBD Downregulated TLR4-Mediated PI3K/Akt and NF-κB/p65
Pathways In Vivo. TLR4-mediated PI3K/Akt and NF-κB pathways play a vital role in regulating inflammatory responses. erefore, this study evaluated the protein expression in the pathways using WB. In vivo, the expression of TLR4 increased significantly in the DSS group compared with the control group, as well as the elevated expression of p-PI3K, p-Akt, p-NF-κB/p65, and p-IκBα (P < 0.01; Figures 5(b)-5(g)). However, the expression of these proteins decreased in the YFBD (10, 20 g/kg) and SASP groups compared with the DSS group (P < 0.05 or 0.01; Figures 5(b)-5(g)).
To explore whether TLR4 is involved in the inhibition of YFBD on PI3K/Akt and NF-κB pathways, we applied siRNA to knock down TLR4 in RAW 264.7 cells and detected the related proteins in the pathways. In this study, TLR4 was remarkably knocked down in siRNA transfected cells (Figure 7(a)). Meanwhile, TLR4-knockdown significantly downregulated the TLR4 expression in LPS-induced RAW 264.7 cells (P < 0.01; Figure 7(c)), and the additional YFBD treatment further reduced the TLR4 expression (P < 0.05; Figure 7(c)). Consistently, the phosphorylation of PI3K, Akt, NF-κB/p65, and the degradation of IκBα increased in LPSinduced cells, whereas TLR4-knockdown attenuated their phosphorylation or degradation (P < 0.01; Figures 7(d)-7(h)), which was similar to the effect of YFBD. Compared with the LPS-induced cells knocked down by siTLR4, the combination of YFBD and TLR4-knockdown decreased the expression of p-PI3K, p-Akt, p-NF-κB/p65, and p-IκBα to a greater extent (P < 0.05 or 0.01; Figures 7(d)-7(h)). is suggests that YFBD acts as an inhibitor of the pathway. Compared with the LPSinduced cells treated with YFBD, the additional interference of siTLR4 decreased the TLR4 expression and the protein expression in PI3K/Akt and NF-κB pathways to the same extent (P < 0.01; Figures 7(c)-7(h)). e results indicated that the effects of YFBD on the inhibition of PI3K/Akt and NF-κB pathways were at least partly through TLR4.

Discussion
UC is threatening the health of people all over the world due to its increasing incidence, undefined etiology, intractability, and recurrence [34]. TCM provides a wealth of weapons against this recurrent disease.
is study confirmed the anti-inflammatory effects of YFBD on DSSinduced models, revealing initially that YFBD exerted its effects partly by regulating TLR4-mediated PI3K/Akt and NF-κB pathways.
DSS-induced colitis closely imitates human UC in terms of both clinical manifestations and anatomical alterations  Evidence-Based Complementary and Alternative Medicine [35]. erefore, the model was used in the study. e results showed that YFBD could mitigate clinical symptoms and pathological changes in DSS-induced models. YFBD, as other prescriptions of TCM, is characterized by complex composition and action on multiple targets. HPLC analysis identified five effective constituents in YFBD: chlorogenic acid, caffeic acid, benzoylmesaconine, benzoyl aconitine, and quercetin. Most of these ingredients showed protective effects against experimental colitis [36][37][38], which cohered with the discovery of the current research. Furthermore, it was shown that high-dose YFBD had more superior curative effects compared with low-dose YFBD. On the one hand, low-dose YFBD could not protect against the invasion of DSS. On the other hand, when the low-dose group was exposed to worse colon injury, the blood flow tended to decrease, accompanied by the reduction in self-repairment ability. e two factors contributed to the significant discrepancy in curative effects between the two groups. Evidence-Based Complementary and Alternative Medicine e mass accumulation of IL-6, IL-1β and TNF-α is one of the features of DSS-induced colitis, which amplifies the inflammatory cascades and accelerates the disease progression [39][40][41].
e serum levels of IL-6 are positively related to the clinical and histopathological severity of UC and can predict the possibility of clinical remission and relapse [42]. IL-1β can activate other cytokine targets, inducing inflammatory response synergistically [43]. e levels of IL-1β in tissues correlated with the disease activity of UC [44]. TNF-α plays a crucial role in the inflammatory response, and anti-TNF-α therapy has been shown as an effective approach [45]. Accordingly, the blocking of these cytokines is considered to be an effective strategy for treating UC. erefore, the study further explored the influence of YFBD on maintaining the levels of inflammatory cytokines. e study revealed that YFBD decreased the levels of IL-1β, IL-6, and TNF-α both in vivo and in vitro. Interestingly, previous studies showed that the active ingredients of YFBD, including caffeic acid, chlorogenic acid, and quercetin, could also downregulate the levels of these cytokines in the colitis models [37,38,46,47], which was in line with the present findings. Although the exact components of YFBD have not been identified, it is presumed that YFBD could achieve remarkable anti-inflammatory effects due to the combination of these ingredients.
Based on the aforementioned results, the study further explored the intracellular mechanism behind the anti-inflammatory properties of YFBD (Figure 8). DSS can destroy the mucosal barrier, which allows bacteria to invade the otherwise impermeable mucus [48]. LPS can be recognized by TLR4 [19,20], and then, the TLR4-linked signaling cascade is initiated, including PI3K/Akt and NF-κB pathways, which are related to inflammatory responses. NF-κB can regulate the gene expression of several proinflammatory cytokines involved in UC pathogenesis [49]. Once activated, NF-κB translocates to the nucleus and triggers the expression of various inflammatory genes. e PI3K/Akt pathway exerts its anti-inflammation effects through regulating the downstream molecules, such as m-TOR, which plays a central role in autophagy induction [50]. A previous study showed that activated m-TOR and impaired autophagy occupied a vital position in intestinal inflammation [51]. Meanwhile, the PI3K/Akt pathway can also enhance the production of proinflammatory cytokines by triggering the NF-κB pathway. is study found that TLR4 was highly expressed in both animal and cell models, accompanied by increased expression of phosphorylated PI3K, Akt, NF-κB/ p65, and IκBα. However, TLR4-mediated PI3K/Akt and NF-κB pathways were downregulated with the treatment of YFBD. Meanwhile, the combination of YFBD and TLR4knockdown decreased the expression of p-PI3K, p-Akt, p-NF-κB/p65, and p-IκBα to a greater extent compared with the LPS-induced cells knocked down by siTLR4 only. A previous research based on network pharmacology indicated that YFBD had positive effects on malignant tumors partly through PI3K and TNF pathways, which strengthened the evidence that YFBD could act on the pathways in our study [52]. ese results demonstrated that the anti-inflammatory activity of YFBD was probably mediated by the inactivation of TLR4-mediated PI3K/Akt and NF-κB signaling pathways and YFBD might play the role of TLR4 inhibitor in treating UC.
However, this study had limitations. First, the signaling pathways related to UC are multiple, and YFBD might have multiple targets in treating UC. Apart from TLR4-mediated PI3K/Akt and NF-κB pathways, other pathways might participate in treating UC simultaneously, which were not examined in this study. Hence, future studies need to explore and clarify the involvement of the other pathways. Second, the components in YFBD are multiple and complex, and the active components involved in treating UC remain unclear. Next, we plan to extract the main active ingredients in YFBD and investigate the effects of these ingredients in treating UC through further experiments.

Conclusions
In summary, YFBD decreased the production of inflammatory cytokines in DSS-induced colitis and LPS-stimulated RAW 264.7 cells. It exerted anti-inflammatory effects via inactivating TLR4-mediated PI3K/Akt and NF-κB pathways.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Disclosure
Liping Chen and Chunyu Zhang are regarded as co-first authors.

Conflicts of Interest
e authors declare that they have no conflicts of interest.